In contrast to the well studied role of intrinsic factors in tumorigenesis, knowledge regarding the mechanism(s) whereby extrinsic factors, such as the stromal/matrix microenvironment, influence an initiated cell with permanent genetic alterations to transform into a tumor is largely unknown. Identifying these key cell signaling events that regulate initiated cells during the tumor promotion stage is an attractive target for designing preventive agents, because initiated cells unlike malignant cells can remain dormant for an extended period of time and are genetically stable, resulting in decreased drug resistance compared with genetically unstable transformed cells. Our long term goals are to define the rate-limiting factors in the matrix microenvironment that affect the survival and/or proliferation of initiated cells. We have identified one of these critical stromal/matrix elements to be osteopontin (OPN), which is a key factor in regulating the survival of initiated cells. We hypothesize that OPN, which is induced during initiation and further induced during tumor promotion, functions in tumorigenesis by preventing apoptosis of transforming keratinocytes (KC) directly through cell surface receptor interaction and/or indirectly by inhibiting the autocrine production of nitric oxide (NO) mediated through suppression of nitric oxide synthase(NOS) production. We define transforming KCs as initiated KCs, which subsequently undergo transformation, and development into papillomas. Our in vivo endpoint of this transforming process is the papilloma, which is readily measured. Specifically, within the two years of funding, we will perform in vitro studies and short-term in vivo studies to 1) define whether OPN functions in the early tumor promotion stages by increasing the proliferation of or by preventing the apoptosis of transforming KC, and 2) elucidate whether OPN-mediated signaling induced by tumor promoters decreases NOS levels and hence NO production. These proposed in vivo and in vitro studies will shed light on the molecular mechanism(s) by which a matricellular protein enhances the survival of transforming cells during early tumor promotion.